For molecular biologists, degradation of nucleic acids using nucleases is particularly important, and is widely used in analytical and preparative methods. In particular, ribonucleases (RNases) are used to degrade ribonucleic acid (RNA) in DNA purification and RNA protection assays. RNase-mediated cleavage of the RNA sugar-phosphate backbone is by transphosphorylation followed by hydrolysis of the 2',3'-cyclic phosphate intermediate formed. The specificity with which an RNase cleaves an RNA molecule is determined by the linear arrangement, or sequence, of purines (adenine, guanine, and, rarely, inosine) and pyrimidines (cytosine and uracil) in the RNA molecule. Virtually all of the well-characterized pancreatic RNase A molecules catalyze an endonucleolytic cleavage after the 3' phosphate group of pyrimidine residues, yet cannot cleave after purine residues. However, since there is little, if any, predictability to the sequences of RNA molecules, a ribonuclease may degrade pyrimidine-rich portions of an RNA to single ribonucleotides, yet leave relatively long pieces of the molecule intact and undergraded. Undergraded oligomers are undesirable by-products in a reaction mixture since they may be long enough to contaminate subsequent reactions by, for example, hybridizing to nucleic acids of interest. It would be desirable, therefore, to provide an RNase that retains its catalytic ability but which also has an ability to catalyze cleavage reactions after residues other than pyrimidines. Such an RNase would be useful for degrading RNA polymers into smaller pieces than are now possible.
Bovine pancreatic ribonuclease A (RNase A; E.C.3.1.27.5) has been an exemplar for studies in all aspects of protein chemistry and enzymology. Because bovine pancreatic RNase A is abundant and relatively easily obtained in purified form, it is considered to be an RNase of choice for use in biotechnology. RNase A is a relatively small protein (14 kDa) that catalyzes the cleavage of the P--O.sub.5, bond of RNA specifically on the 3'-side of pyrimidine nucleosides, by a two step mechanism in which a cyclic phosphate intermediate is formed. Other RNase A's that have been analyzed are quite similar in sequence and specificity to the bovine pancreatic form.
It has been inferred from structural analysis of RNase A that the sidechain of the threonine residue at position 45 (Thr45) mediates the pyrimidine specificity by forming hydrogen bonds with a pyrimidine base (U or C), and by sterically excluding the purine bases (A, G, or I). The structural data also show that the aromatic sidechain of the phenylalanine at position 120 (Phe120) stacks with the base of pyrimidine residues.
RNase A forms a three dimensional structure having at least three subsites (B1, B2, B3) that each bind to a base in polymeric RNA. Thr45 and Phe120 contribute to the B1 subsite, which is highly specific for pyrimidine bases as discussed above. Subsite B2 binds to the base 3' to the pyrimidine base in B1 and subsite B3 binds to the base 3' to the base in B2. While subsites B2 and B3 are able to accept all bases, B2 has a 100-fold preference for adenine and B3 has a 10-fold preference for adenine.
In addition to a desire to broaden the number of bases at which RNase A can cleave RNA, it would also be desirable to provide a processive RNase A that could repeatedly catalyze cleavages along the length of a single RNA polymer until every possible cleavage of the polymer has occurred. Wild-type RNase A, in contrast, is a distributive enzyme, in that after binding to an RNA polymer, the RNase A molecule catalyzes a single cleavage reaction then dissociates from the cleaved polymer. Thus, each cleavage requires a separate interaction between enzyme and substrate. A naturally occurring processive RNase is RNase II, a cytosolic enzyme from E. coli.
To facilitate more complete degradation of undesired RNA, molecular biologists overcome the pyrimidine specificity of pancreatic RNases by mixing a pancreatic RNase such as RNase A with RNase T1, which cleaves RNA after G residues, to form a cocktail that cleaves RNA at three of the four residues. Yet, even this approach does not ensure complete degradation of every RNA species in a reaction vessel, since phosphodiester bonds after A residues are still not cleaved.
Other solutions to the problem of incomplete RNA digestion exist. A 27 kDa periplasmic enzyme, recently cloned from E. coli and overproduced, cleaves the phosphodiester bond between all four nucleotide residues. Meador, J. et al., 187 Eur. J. Biochem. 549 (1990). However, apparently this enzyme is relatively unstable and therefore difficult to handle without rendering it inactive.